WO1992017151A2 - Procede d'administration de medicaments par l'intermediaire de la modification des jonctions occlusives eptheliales par le facteur de necrose tumorale - Google Patents

Procede d'administration de medicaments par l'intermediaire de la modification des jonctions occlusives eptheliales par le facteur de necrose tumorale Download PDF

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Publication number
WO1992017151A2
WO1992017151A2 PCT/US1992/002312 US9202312W WO9217151A2 WO 1992017151 A2 WO1992017151 A2 WO 1992017151A2 US 9202312 W US9202312 W US 9202312W WO 9217151 A2 WO9217151 A2 WO 9217151A2
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epithelial
pharmacologically
tnf
active
resistance
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PCT/US1992/002312
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WO1992017151A3 (fr
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James M. Mullin
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Lankenau Medical Research Center
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Publication of WO1992017151A2 publication Critical patent/WO1992017151A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta

Definitions

  • This invention relates to improved methods and systems for administering pharmacologically-active agents by transepithelial (paracellular) administration techniques. More specifically, the transepithelial or paracellular administra ⁇ tion of pharmacologically-active substances may be accomplished through the prior administration of biologically-active substances that interact with the receptors for the cyto ine Tumor Necrosis Factor (the serum factor inducing hemorrhagic necrosis of transplantable tumors; "TNF”; also known as the macrophage hormone associated with cachexia or "cachectin”) located on epithelial membranes, causing a reversible dis ⁇ ruption of the tight junctional strands between such epithelial cells, thereby promoting the passage of pharmacologically- active substances and solutes through the intercellular voids caused by such disruption of the tight junction between epi ⁇ thelial cells.
  • TNF tumor Necrosis Factor
  • the method is most significant with respect to the administration of higher molecular weight peptides, pro ⁇ teins, and the like, specifically the ability of peptides and proteins of interest to transit epithelial barriers and enter the blood stream without undergoing degradation (as would occur if they transited through - not between cells of the barrier) .
  • the systems of the invention involve the sequential administra ⁇ tion of a biologically-active substance reactive with the TNF receptor followed by a pharmacologically active substance in a pharmaceutically acceptable carrier.
  • TPA 12-0-tetradecan- oyllphorbol-13-acetate
  • PDBU phorbol-12,13-dibutyrate
  • Epithelial tight junction disrupting substances such as exogenously administered TPA and other phorbol esters have been shown to act when administered to either the apical (luminal) or basal-lateral (antiluminal) cellular surfaces.
  • Transepithelial flux of D-mannitol and polyethylene glycol following the application of phorbol ester tumor-promoting agents to LLC-PKi renal epithelial cells has demonstrated that the normally occluding junctions between cells have been made leaky.
  • EGF epidermal growth factor
  • PDBU phorbol ester
  • Other compounds shown to produce enhanced trans ⁇ epithelial permeability include certain diacylglycerol com ⁇ pounds, such as 1,2-dioctanoylglycerol, 1,2-dicaprylglycerol, 1,1-dioleoylglycerol or l-oleoyl-2-acetylglycerol.
  • these agents also are activators of the protein kina ⁇ e C signal transduction system, as are phorbol esters, it has been sug ⁇ gested that the changes in epithelial junctional permeability may, at least in part, be mediated through such a mechanism.
  • the regulation of transepithelial permeability through protein C kinase activation has been suggested as a mean ⁇ of promoting the transit of protein hormones across the blood-brain barrier or the chordus-plexus epithelia.
  • the diacylglycerol compounds and, in particu ⁇ lar, 1,1-dioctanoyl glycerol show some reversibility of the initial dissipation of transepithelial resistance.
  • the diacyglycerols are endogenous compounds that would not under normal circumstances be accessible to the TNF receptors located on the cellular membranes of epithelial cells, and moreover have (as "intracellular second messengers") extremely wide ranging effects. In addition their action on cell junc ⁇ tions is also not rapidly reversible.
  • various biologically-active substances may elicit changes in the homeo ⁇ static tight junctional resistance of epithelial cell sheets to varying degrees.
  • the biologically-active agents known to affect the tight junctional barrier suffer various drawbacks of irreversibility, the need for replenishment of said agent to maintain a reduced tight junctional resistance level, the incomplete reversion to the homeostatic resistance level following administration of said agent, and a lack of potency.
  • the phorbol esters and diacylglycerols suffer a severe drawback relating to their suspected status as car ⁇ cinogens and/or agents having plasma membrane disruptive activity.
  • An object of the present invention is to provide a method of administering pharmacologically-active substances which normally present absorption problems across epithelial membranes due to the structure, molecular weight, solubility, degradation or other physico-chemical problems associated with the absorption of such agents.
  • Another object of the present invention is to provide a system of administering pharmacologically-active substances which normally present absorption problems across epithelial membranes due to the structure, molecular weight, solubility, degradation or other physico-chemical problems associated with the absorption of such agents.
  • Yet another object of the present invention is to provide a method of sequentially lowering the tight junction resistance of epithelial cells below homeostatic levels to permit paracellular transport of exogenously administered pharmacologically-active substances.
  • a method is provided of transepithelially administering (by paracellular route) pharmacologically-active substances, preferably peptide, protein and other pharmacologically-active substances that normally present absorption problems across mammalian epithelial membranes, and most preferably, peptides and other pharmacologically-active substances of a molecular weight of about 6,000 or less, comprising:
  • a pharmacologically-active substance in a pharmaceutically-acceptable carrier (for example water or other pharmaceutically acceptable diluents or carriers for said pharmacologically-active substance) to said predetermined mammalian epithelial cells before said tight junctional re ⁇ i ⁇ - tance reverts to said ho eostasic state, preferably within about 60 to about 150 minutes following the administration of said biologically-active substance reactive with said receptor for tumor necrosis factor.
  • a pharmaceutically-acceptable carrier for example water or other pharmaceutically acceptable diluents or carriers for said pharmacologically-active substance
  • Another aspect of the present invention discloses a method of paracellularly administering pharmacologically-active substances across mammalian epithelial membranes, comprising:
  • transepithelial therapeutic system containing a pharmaceutic composition for transepithelial administration of pharmacologically-active substances to predetermined epi ⁇ thelial tissue, comprising:
  • a tight junction resistance-lowering amount of tumor necrosis factor preferably TNF-alpha
  • a pharmaceu ⁇ tically acceptable carrier which enhances the transepithelial permeability of component (b) ;
  • a pharmacologically-active substance preferably peptides, proteins and the like and most preferably compounds of a molecular weight of le ⁇ than 6,000 in a pharmaceutically acceptable carrier (preferably water) .
  • Figure 1 i ⁇ a graph depicting the dose response of TNF on transepithelial resistance.
  • Figure 2 is a graph depicting the delayed enhancement of transepithelial resistance by TNF.
  • Figure 3 is a graph depicting the effect of apical vs. basolateral TNF on transepithelial resistance.
  • Figure 4 i ⁇ a graph depicting the transepithelial resistance decrease induced by TNF.
  • Figure 5 is a graph depicting the effect of TNF on LLC-PK ! transepithelial resistance: the 4 phases of the re ⁇ sponse.
  • Figure 6 is a graph depicting the effect of TNF on LLC-PK- transepithelial resistance following pretreatment with genistein, to block tyrosine kinase.
  • Figure 7 is a graph of TNF on LLC-PKi transepithelial resi ⁇ tance: pretreatment with cycloheximide to block protein ⁇ ynthe ⁇ i ⁇ .
  • Figure 8 is a graph of lack of tight junctional res- pon ⁇ e upon secondary TNF application.
  • epithelial cells i ⁇ deemed to include epithelial as well as endothelial cells lining the blood and lymph vasculature and other closed bodily cavities such as the heart and tho ⁇ e capillarie ⁇ forming the blood-brain barrier.
  • the terms “paracellularly” and “tran ⁇ epithelially” are intended to connote transit of materials or solutes around and between epithelial cells, rather than through intracellular absorption of such materials and solutes.
  • the terms “tight junctional barrier,” “tight junctional region” or “tight junction” are known in the art to signify the ring ⁇ like junctional seal between mammalian epithelial cells which line body cavities.
  • the term "homeo ⁇ ta ⁇ i ⁇ ” or “homeo ⁇ tatic” i ⁇ intended to connote the normal dynamic equilibrium ⁇ tate of the tight junctional resistance of mammalian epithelial cells. "Sequential application" of the TNF-receptor activator and the pharmacologically active substance may potentially encompass a negligible time difference to one of several hours, reflecting the different physiology of different epithelial barriers and different species, which selection may be made by one of ordinary skill in the art without undue experimentation.
  • epithelial cells of the human body An essential function of epithelial cells of the human body is to divide the body into separate fluid-filled compartments, delimited by these epithelial (or endothelial) cells.
  • the cell ⁇ dividing these compartments perform, beyond their essential barrier function, thermodynamic work by trans ⁇ porting solutes unidirectionally from one fluid compartment to the other, thereby defining and maintaining the unique compo ⁇ sition of these compartments.
  • the cells of the epithelial or endothelial barrier are, however, only one component of the barrier. Equally important is the junctional band surrounding each cell of the cell sheet.
  • This "tight junction" or zonula occluden ⁇ is a semipermeable barrier which allows certain solutes (depending upon their size and charge) to pass through this paracellular pathway between the cells from one fluid compartment to the other.
  • the tight junction is fully distinct from the zonula adherens, adhering junction, or "desmosome” which presents no barrier to solute flow.
  • the tight junction is composed of a network of protein strands, the composition of which is just beginning to be understood. It is believed that the tight junction is not static, but appears to be highly regulated, producing permeability changes in response to a large number of changing physiological conditions. Accord ⁇ ingly, it is deemed to play a key role in transepithelial transport processes previously thought to occur through the cell ⁇ per ⁇ e.
  • TNF tumor necrosis factor
  • exogenou ⁇ administration of TNF may be targeted to react with TNF re ⁇ ceptors on epithelial cell ⁇ of a predetermined epithelial membrane and may be therapeutically employed to modify the occlusion of epithelial cells to facilitate the transit of solutes including normally problematic pharmacologically-active substances, as aforesaid.
  • TNF is but one member of a family of cytokine ⁇ relea ⁇ ed by cell ⁇ of the immune system in response to various invasive stimuli.
  • TNF-alpha has been shown to be the mo ⁇ t active form of the cytokine and i ⁇ ⁇ pecified for u ⁇ e in the preferred embodiment of the invention and i ⁇ deemed to be the be ⁇ t mode of practicing the invention.
  • TNF-beta exhibit ⁇ ⁇ ignificantly le ⁇ activity.
  • an LLC-PKi renal epithelial cell line wa ⁇ cho ⁇ en becau ⁇ e of it ⁇ clo ⁇ e parallel in phy ⁇ iological properties to the proximal tubule of the kidney, one of the most exten ⁇ ively inve ⁇ tigated epithelial preparations.
  • TNF may be administered as a single application to an epithelial membrane, whereupon once in contact with TNF, the affected cell membranes will lower the resistance of the tight junctional barrier, allowing the passage of additional solutes including, for example, exogenously administered pharmacologically-actice substances.
  • Fig. 2 exemplifie ⁇ the normal occurrence that re ⁇ i ⁇ - tance not only return ⁇ to original value ⁇ but also normally rises above control values. This "overshoot" is transient and resistance values return to initial levels usually before 12 hours. The observed gradual decrease in control resistance levels i ⁇ characteristic of my experiments. It is typically a slow, graded decrease which rarely falls below 80% of initial resistance values.
  • D-["C]mannitol was added to the baso- lateral fluid compartment and then the rate of appearance of D["C]-mannitol in the apical compartment wa ⁇ measured. It is under ⁇ tood in the art that D-mannitol has negligible affinity for any membrane transport system in LLC-PK X cell ⁇ and must cross the cell sheets by passing across the tight junctions and moving between the cells.
  • Average resistance of three cell sheets ⁇ range of values. b Average of linear regression determinations for three cells sheets ⁇ SE.
  • the drop in resistance was of equal magnitude and identical time course in each case.
  • TNF monomer ⁇ have molecular weight ⁇ of 17,000 and the active form of TNF may be a trimer with a molecular weight of 55,000.
  • Thi ⁇ signifies TNF would be active in this effect from within a luminal compartment.
  • TNF was preincubated with a monoclonal antibody to TNF for 30 minutes at 25°C in culture medium. This resulted in a complete inhibition of the effect of TNF on tight junctional resistance (See Fig. 4) .
  • the monoclonal antibody produced no effect on transpithelial re ⁇ i ⁇ tance.
  • ⁇ ub-optimal level ⁇ of TNF would be without effect and (2) repeated expo ⁇ ure to TNF i ⁇ without ⁇ econdary effect (See Figure 8) .
  • tyrosine kinase inhibitor genis- tein
  • the tyrosine kinase inhibitor is to date the only compound which I have tested which can block (dose dependently) the actual effect, i.e. the increase in junction permeability (the decrease in trans ⁇ epithelial resistance) (see Figure 6) .
  • An epithelial barrier will not stay chronically "open” if TNF i ⁇ in continual contact, or if TNF i ⁇ readminis- tered after a fixed period of time. This means that the physiological side effects should be minimized as e.g. residual administered TNF cannot keep the barrier leaky.
  • TNF action on epithelial barriers appears to be capable of being fully blocked by inhibiting tyrosine kinase.
  • Thi ⁇ may allow ⁇ electivity in tissue specific action.
  • TNF effect on epithelial barriers will not be reversible if the ability of the cells to synthesize new protein is compromised. This signifies that use of the TNF receptor a ⁇ a means of drug delivery must preclude the simul ⁇ taneous use of any drug ⁇ which would impair protein synthesis.
  • LLC-PK pig kidney epithelial cells used in these studies are from passages 185 to 200. Routine culturing entailed seeding 1 X 10 5 cells in a 75-cm z culture dish con ⁇ taining 25 ml of alpha-minimum essential medium with 10% fetal bovine serum. After 1 week at 37°C and 5% C0 2 , the culture reached confluence, it wa ⁇ then trypsinized, and the passaging was repeated.
  • a trypsinized suspension was seeded into Millicell HA future cup assemblies (Millipore Corp.) containing 2 ml of culture medium with 10% fetal bovine serum and incubated at 37°C in a 5% C0 2 humidified atmosphere.
  • the ⁇ e 30mm-diameter a ⁇ emblies had a filter base with 0.45-um pore ⁇ .
  • Three such filter-cup assemblie ⁇ were placed in a 100-mm Petri di ⁇ h containing 15 ml of culture medium. When cell ⁇ formed a confluent monolayer, an intact epithelium exists across the filter base.
  • cell sheets in filter-cup assemblies were refed with normal medium or medium containing TNF. Resistance values were measured prior to the above refeeding and then at 0.5-hour intervals. After 2 hour ⁇ at 37"C, resistance values were maximally decreased.
  • the control set (of three cell sheets) and one set of TNF-treated cell sheets were then rinsed three time ⁇ in (morpholinopropanesulfonic acid-buffered) saline at 25 ⁇ C.
  • the culture medium used in these ⁇ tudie ⁇ is a product of Hazelton Research Products.
  • the fetal bovine serum was purchased from Hy-Clone Laboratories, Inc.
  • the human recom- binant tumor necrosis factor-alpha wa ⁇ obtained either from Boehringer Mannheim [2.2 X 10 7 unit ⁇ /mg protein (determined by a cell lytic a ⁇ say with L929 cultures)] or Genentech [4.3 X 10 7 units/mg protein] .
  • the anti-TNF-alpha monoclonal antibody wa ⁇ a product of Boehringer Mannheim.
  • the D-[ 14 C] mannitol u ⁇ ed in the tracer flux study was obtained from ICN Radiochemical ⁇ , Inc.
  • Cell ⁇ heet ⁇ in filter-cup assemblies were refed with normal medium or medium containing 40 ng/ml TNF. Resistance values were measured at 0.5-hour interval ⁇ , until maximally de ⁇ creased (2 hour ⁇ ) , and then cell sheets (Sets A and B) were rinsed in saline and a flux experiment was performed as de ⁇ scribed above. The rate of D-mannitol flux was then determined by linear regression of the plots of appearance of D-["C] mannitol versus time. The third set of cell sheets (Set C) wa ⁇ incubated for 3 hours at 37 ⁇ C, by which time resistance re ⁇ covered to 72% of the initial readings. The above flux proce ⁇ dure wa ⁇ then performed on these cell sheets. The results are reported above in Table 1.
  • the transepithelial resistance across pairs of LLC-PK cell ⁇ heet ⁇ wa ⁇ mea ⁇ ured following the general proce- dure ⁇ outlined above.
  • one set of cell sheet ⁇ (control) in filter-cup a ⁇ emblies wa ⁇ refed with normal medium.
  • the other ⁇ et wa ⁇ refed with medium containing 10 ng/ml TNF.
  • the readings of two cell sheets for each condition are reported here.
  • the control resistances show a slight (15%) but steady decline of resistance values during the course of the experiment.
  • Cell ⁇ heet ⁇ treated with TNF have the charac ⁇ teristic reversible resistance drop at approximately 2 hours. After the recovery, resistance values not only rise above control levels but eventually exceed initial level ⁇ before slowly returning to control levels. Results of this experiment are presented graphically in Fig. 2.
  • Re ⁇ ult ⁇ are expre ⁇ ed a ⁇ the average of three cell sheet ⁇ (percertage of initial resistance) ⁇ SE.
  • Cell sheet ⁇ incubated with antibody but without TNF-alpha showed a tracing very similar to that of control.

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Abstract

Procédé et système d'administration trans-épithéliale de substances pharmacologiquement actives à travers les barrières épithéliales mammifères. Ils comportent l'administration séquentielle à des cellules épithéliales prédéterminées comprises dans la barrière de cellules épithéliales mammifères, portant des récepteurs du facteur de nécrose tumorale et présentant entre elles une résistance homéostatique de jonction occlusive, d'une quantité suffisante d'une substance biologiquement active apte à entrer en réaction avec lesdits récepteurs du facteur de nécrose tumorale, pour réduire ladite résistance homéostatique de jonction occlusive et pour permettre ainsi le passage trans-épithélial desdites substances pharmacologiquement actives à travers lesdites barrières; puis l'administration exogène d'une substance pharmacologiquement active dans un vecteur pharmaceutiquement acceptable à une barrière épithéliale mammifère prédéterminée avant que ladite résistance de jonction occlusive ne retourne audit état homéostatique.
PCT/US1992/002312 1991-03-27 1992-03-20 Procede d'administration de medicaments par l'intermediaire de la modification des jonctions occlusives eptheliales par le facteur de necrose tumorale WO1992017151A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0696456A2 (fr) * 1994-05-20 1996-02-14 BEHRINGWERKE Aktiengesellschaft Combinaison de substances induisant de la nécrose avec des substances qui sont activées par la nécrose pour le traitement sélectif de tumeurs et de maladies inflammatoires

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989004663A1 (fr) * 1987-11-18 1989-06-01 State Of Oregon Acting By And Through The State Bo Procede d'administration de matiere genetique a travers la barriere sanguine du cerveau

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989004663A1 (fr) * 1987-11-18 1989-06-01 State Of Oregon Acting By And Through The State Bo Procede d'administration de matiere genetique a travers la barriere sanguine du cerveau

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Cancer Research, vol. 50, no. 7, 1 April 1990, J.M. MULLIN et al.: "Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability", pages 2172-2176, see the whole document *
FEBS Letters, vol. 221, no. 2, 14 September 1987, J.M. MULLIN et al.: "The phorbol ester, TPA, increases transepithelial epidermal growth factor flux", pages 359-364, see the whole document *
J. Cell. Biology, vol. 115 (3 part 2), 1991, A.D. MARMORSTEIN et al.: "Tumor necrosis factor but not interleukin-1 increases the permaebility of a renal epithelium (MDCK)", page 480A, see the whole document *
The Journal of Cell Biology, vol. 109, no. 4, October 1989, J.M. MULLIN et al.: "Tumor necrosis factor causes leakiness in epithelial tight junctions", see the whole document *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0696456A2 (fr) * 1994-05-20 1996-02-14 BEHRINGWERKE Aktiengesellschaft Combinaison de substances induisant de la nécrose avec des substances qui sont activées par la nécrose pour le traitement sélectif de tumeurs et de maladies inflammatoires
EP0696456A3 (fr) * 1994-05-20 1998-10-28 BEHRINGWERKE Aktiengesellschaft Combinaison de substances induisant de la nécrose avec des substances qui sont activées par la nécrose pour le traitement sélectif de tumeurs et de maladies inflammatoires

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